Hypoxia is known to confer resistance of cancer cells to radio- and chemo-therapies leading to treatment failure. Our long-range goal is to induce apoptosis of hypoxic cancer cells through targeting the molecules that confer such a resistance. SAG (Sensitive to Apoptosis Gene) or ROC2/Rbx2 is a cysteine-rich antioxidant protein and a RING component of SCF (skp1, Cullins, F-box proteins) and possibly VCB (VHL-Cul2-Elongin B and C) E3 ubiquitin ligases. We found that SAG is induced under hypoxia and when over-expressed, inhibits hypoxiainduced apoptosis both in vitro and in vivo. SAG also binds to caspase 3 and promotes caspase 3 ubiquitination when complexed with SCF E3 ubiquitin ligase. The objective of this application is to elucidate the mechanism of SAG induction and apoptosis protection under hypoxia. The central hypothesis is that hypoxia induces HIF-1 which transactivates SAG expression through a direct binding to its consensus elements in the SAG promoter. Hypoxia also induces activity of protein kinase CKII which activates SAG via phosphorylation. Upon its induction and activation, SAG complexes with other components of SCF and VCB E3 ubiquitin ligases to promote the ubiquitination and degradation of caspase 3 and HIF-1alpha, respectively. Targeted caspase 3 degradation blocks caspase-dependent apoptosis, whereas targeted HIF-1alpha degradation prevents HIF-1alpha induced apoptosis through p53 stabilization. The rationale for the proposed research is to identify and characterize SAG and CKII as novel hypoxic cancer targets with ultimate goal in pharmaceutical intervention through their inhibition to sensitize hypoxic cancer cells to current cancer therapies. Specific aims to test the hypothesis are 1) to elucidate the mechanism of SAG induction through HIF-1alpha transcriptional activation under hypoxia, 2) to elucidate the mechanism by which SAG protects against apoptosis in manners of ligase independence (scavenging reactive oxygen species) and ligase dependence (caspase 3 and HIF-1alpha ubiquitination and degradation), 3) to determine the sensitization of hypoxic cancer cells to radio-therapy through targeting SAG and CKII, and 4) to differentiate SAG from ROC1/Rbx1 in their stress responses. Through this proposed research, we will demonstrate, for the first time, a feedback loop between SAG and HIF-1alpha and reveal a mechanism by which SAG inhibits apoptosis under hypoxia via promoting the ubiquitination/degradation of caspase 3 and HIF-1alpha. We will also demonstrate a radio-sensitizing effect achieved by SAG silencing and CKII inhibition and provide a molecular basis for future discovery of novel radio-sensitizers through targeting SAG, or SAG-associated E3 ligases, and/or CKII kinase.